We plan to continue our detailed analysis of metants of the alphavarius, Sindbis virus. We now have almost 200 mutants of the virus, most of which have been characterized for RNA synthesis at the nonpermissive temperature, pattern of proteins produced in the infected cell, and complementation group. Of particular interest are certain well characterized RNA+ mutants, including some which insert membrane glycoproteins into the endoplasmic reticulum at the nonpermissive temperature but fail to transport them to the plasma membrane, some which lead to an altered intereaction between the capsid and the glycoproteins during virus maturation and mutants which alter the processing of precursor polypeptides. We are also interested in the large plaque to small plaque mutation which affects host preferences and response to the ionic environment as well as plaque morphology. We propose to identify the particular nucleotide (and thus amino acid) changes in a number of these mutants by direct chemical sequencing of selected regions of the cDNA made from the 26S mRNA for the structural proteins isolated from infected cells. We have recently obtained the entire nucleotide isolated from infected cells. We have recently obtained the entire nucleotide sequence of the 26S mRNA of the HR parent of these mutants using this method and can rapidly extend this to the mutants. Changes in selected mutants will be confirmed by microsequenation of proteins. By mapping the sites of mutation in this way and correlating the effects of amino acid substitutions of deletions with specific physiological defects, we hope to identify the functional domains of the virus proteins. We plan to raise specific antibodies to the non-structural proteins produced during Sindbis infection by chemically synthesizing peptides corresponding to nucleotide sequences from the 49S genomic RNA, and using them as antigens after coupling to appropriate carriers. By specific immunoprecipitation with these antibodies we can study the pattern of synthesis and processing of non-structural proteins made by the RNA- and RNA+ mutants. These studies are important to our understanding of virus replication and assembly and of biosynthesis and transport of integral membrane glycoproteins, and are important because a number of alphaviruses, including several endemic in the U.S., are important health hazards.